Identification of stable internal control genes for accurate normalization of real-time quantitative PCR data in testicular tissue from two breeds of cattle

Abstract

Objective: To assess the stability of 10 candidate internal control genes (ICGs), namely GAPDH, ACTB, RPL23, RPS15A, ATPSF1, GLUT5, HMBS, ATP2B4, PPIA, and BRP to normalize the transcriptional data from testes samples of Zebu and crossbred bulls. Methods: Total RNA was isolated from testicular tissue of Zebu and crossbred bulls (n=6 each) between 2-8 years of age. cDNA was synthesized, and the quantitative real-time polymerase chain reaction (PCR) was performed. The cycle threshold values were used for the analysis of the stability of ICGs. Four different statistical algorithms: geNorm, Normfinder, BestKeeper, and RefFinder, were used to assess the stability of these genes. Results: ATPSF1, HMBS, PPIA, and RPS15A were the most reliable and stable ICGs for Zebu testes, and ATPSF1, RPL23, and PPIA for crossbred testes. Conclusions: A panel of stable ICGs (ATPSF1, HMBS, PPIA, RPS15A for Zebu and ATPSF1, RPL23, and PPIA for crossbred) for normalization of gene expression data in testes samples can be helpful for researchers to conduct functional genomics studies at the testicular level in cattle bulls.

Keywords

• cattle bulls; gene expression; internal control genes; normalization; qpcr; testis